The water soluble vitamin, biotin, plays a critical role in carbohydrate, lipid, and amino acid metabolism. Yet, there is little known concerning its transport and metabolism in animal cells. Defects in these processes may be the primary cause of the reported "biotin responsive" diseases in both humans and animals. This warrants further study of these phenomena. In the proposed investigations biotin metabolism will be studied in mouse 3T3-L1 cells and in isolated rat liver mitochondria. Fibroblasts of the 3T3-L1 cell line will differentiate in culture to cells with adipocyte like characteristics. During this conversion the levels of the biotin enzymes increase dramatically. Research studies are proposed to elucidate the mechanism of transport of biotin into the 3T3-L1 cell and to examine its subsequent intracellular metabolism. The transport process will be characterized with regard to its kinetics, substrate specificity and response to serum components. The relationship between biotin utilization and its catabolism will be assessed. These studies will be carried out with non-differentiated and fully differentiated cells. These experiments should be useful in delineating the factors which regulate intracellular biotin metabolism. Three of the four biotin dependent enzymes in the liver are located within the mitochondria. The significance of the mitochondrial membrane in regulating intracellular biotin utilization is not understood. Research studies are planned to explore the mechanism of biotin transport into isolated rat liver mitochondria. Investigations will be directed transport into isolated rat liver mitochondria. Experiments will be executed to explore the relationship between intramitochondrial biotin uptake and holocarboxylase formation within this organelle.